Compositions containing a tetracycline antibiotic and potentiating agents therefor



United States Patent COMPOSITIONS CONTAINING A TETRACYCLINE ANTIBIOTICAND POTENTIATING AGENTS THEREFOR Joseph M. Pensack, Trenton, N.J., andCharles N. Huhtanen, New Hope, Pa., assignors to American CyanamidCompany, Stamford, Conn., a corporation of Maine No Drawing. Filed Jan.15, 1963, Ser. No. 251,477

4 Claims. (Cl. 167-53) This invention relates to novel compositionscontaining a tetracycline antibiotic and a potentiating agent which arehighly useful in controlling infectious diseases and in stimulating thegrowth of animals. More particularly, the present invention is concernedwith an animal feed composition for non-ruminants, such as poultry andswine, containing a tetracycline antibiotic and a novel group ofadjuvants which promote increased absorption of the antibiotic andassimilation thereof by the blood and tissue of the animal.

Although the broad-spectrum antibiotics chlortetracycline, tetracycline,oxytetracycline, and demethylchlortetracycline have been found to beextremely effective for controlling infectious diseases and forstimulating growth in animals; nevertheless, due to incompleteabsorption thereof by the body of the animal, believed to be due atleast in part to the presence of calcium and other metallic cations inthe diet, f-ull utilization of such antibiotics has never been achieved.Since such antibiotics are relatively expensive, it is most desirable toprovide a means whereby their effectiveness is fully realized.

It is known that the standard sources for calcium and phosphorus inanimal diets, particularly poultry diets, are generally calciumcarbonate, calcium hydrogen phosphate and bone meal. These materials,while generally considered necessary for normal poultry growth, havebeen found to markedly lower the blood levels of the tetracyclineantibiotics. Since improvement in health is normally related to bloodlevels of antibiotic, efforts have been made to overcome the deleteriouseffects of calcium on the blood levels of the antibiotic withouteliminating calcium from the diet.

While compounds such as terephthalic acid have been employed to increaseblood levels of antibiotic, such materials have not been whollyeffective since it has been found that the blood level of antibioticcannot be significantly increased above that which is attained with anoptimum dosage even though the dosage is increased twoor three-fold.

Attempts have also been made to improve blood levels from a given amountof tetracycline antibiotic by employing a low calcium diet, i.e., a dietcontaining well below 0.8% calcium. However, this method has not provedto be entirely satisfactory since a normal chick diet requires about0.8% calcium for normal growth. Thus continuous low calcium diet cannotbe tolerated if normal growth is to be obtained.

A still further attempt to increase blood levels from a given amount oftetracycline antibiotic has been made wherein calcium sulfate isemployed as the sole added source of calcium in the diet. This methodhas met with considerable success since it provides blood levels oftetracycline antibiotic comparable to those obtained with the lowcalcium diet or the feed containing terephthalic acid. However, thisprocess like the others is not entirely satisfactory since there is somewaste involved because the antibiotic is not completely absorbed fromthe alimentary tract. Furthermore, this method of treatment has thedisadvantage that relatively high levels of calcium sulfate tend toproduce a wet litter which is an excellent breeding ground forinfectious microorganisms.

3,282,779 Patented Nov. 1, 1966 It would be most desirable commerciallyto obtain compositions which would permit the effectiveness of thetetracycline antibiotics to be appreciably enhanced. It has been foundthat this most desirable object can be achieved with the novelcompositions of the present invention which comprise a tetracyclineantibiotic, that is,

; higher concentrations in the blood of the animals, with the use of agiven amount of the antibiotic, or conversely resulting in equivalentblood levels with much less of the antibiotic compound than it ispossible to obtain with the antibiotic alone.

The novel potentiating agents of this invention may be administeredsimultaneously with the administration of the tetracycline antibiotic.Generally the quantity of agent is limited. Excellent results have beenobtained with amounts equivalent to between 1 and 5 parts by weight per1,000 parts by weight of the feed. The adjuvants may be employed invarious forms, for example, pills, tablets, capsules, in solution-or infine granules and may be administered in various ways, as by forcefeeding or in the drinking water or feed. It is preferred that i thelatter method of administration be employed since it biotic at minimumcost.

is the easiest method of administration, requires no additional efforton the part of the farmer when the feed is purchased as a premix andprovides a means for obtaining optimum effectiveness of the adjuvantsand the anti- Optimum ratios of adjuvant to antibiotic can be readilycontrolled. In addition to the adjuvant and antibiotic, the feed maycontain nutritionally-balanced quantities of carbohydrates, proteins,vitamins and minerals. If tablets, capsules, pills, pellets or the likeare employed, they may contain in addition to the adjuvant andantibiotic, excipients and diluents such as sugars, starches and thelike.

Any of the commercially available and commonly used tetracyclineantibiotics such as tetracycline, chlotetracycline, oxytetracycline,demethylchlortetracycline, and the like may be used. Chlortetracycline,however, is preferred because of its already wide acceptance as ananimal feed additive of choice and because it is more effective than theother tetracycline antibiotics.

Preferably, the tetracycline antibiotic is present to the extent ofbetween about 50 and 2,000 parts per million based on the weight of thefeed with the optimum amount being about 200-400 parts per million.

The novel potentiating agents of this invention may be represented bythe following general formula:

ROa

R0 I ia wherein R is H or alkali metal; R is H or NH R and R are H, OH,SO H, or SO -alkali metal; R is H, NH SO H, SO -alkali metal,substituted benzoylamino, carbamoyl or (substituted phenyl) carbamoyl,i.e.,

NHC O- NO:

or -N=NZ, wherein Z is a member of the group consisting of phenyl,substituted phenyl, substituted amino, 15

and substituted heterocyclic radicals, i.e.,

I I OH;

I orrfiorr AH l @CI'DOH ONa NHCH;

NH C HzSOaNa and R is H, or substituted phenylazo, i.e.,

In a more preferred embodiment of the present invention it has beenfound that the 5,7-disulphonic compounds of the following formula arepreferred potentiating agents.

wherein R is -H or alkali metal; R is H or NH R is H or OH; R is H orsubstituted benzoylamino, i.e.,

70 R is H, NH OH, NH -HCl,

NH NH NrI-ii-NE- Nm 75 Or N=N-Z, wherein Z is a member of the groupconsisting of phenyl, substituted phenyl and substituted heterocyclicradicals, i.e.,

I NH-CHrN N-CHzOH IIH I @CEHOH O Na I owl-@021 NHCH;

I OCH;

NH CHzSOaNa NH O C H and R is H or phenylaz-o.

and

In a still more preferred embodiment of the present invention it hasbeen found that the compounds having the general formula given belowappear to produce a most remarkable increase in antibiotic blood levelsin animals.

Ilh R2 SO R 1' wherein R is H or alkali metal; R is H or NH R is H orOH; R; is H, OH, NH or -N=NZ, wherein Z is a member of the groupconsisting of phenyl, substitutedphenyl and substituted-heterocyclicradicals, i.e.,

NH-CHz-N N-C HzOH and The following compounds, which have been found tobe useful as potentiating agents, are known to the art and theirpreparation will not be described:

The preparation of other related compounds which have also been found tobe highly effective as potentiators for antibiotics will be described inthe examples which follow.

The invention will be further described in conjunction with thefollowing specific examples.

EXAMPLE 1 Preparation 07 5,7-naphthalenedisalfnic acid, 3-(4-acrylamido-o-tolylazo), disodium salt 5.09 g. of glacial acrylic acidand 7.66 g. of phosphorus oxychloride were added to 300 m1. of drypyridine at such a rate that the temperature of the mixture did notexceed 20 C. Following this addition the temperature of the mixture waslowered to from 0-5 C. At this temperature, 14.53 g. of the azo compoundderived from diazotizing amino G acid and coupling in-to m-toluidine wasadded to the reaction mixture. The temperature was raised to 95 C.andmaintained at this temperature for 3 /2 hours. The mixture was thencooled to room temperature and 500 ml. of water added thereto. Pyridinewas then steam stripped from the mixture. The remaining solution wastreated with sodium chloride at 70 C. to precipitate the product. Theproduct was collected by filtration and washed with 20% brine. Theproduct was then dried at 55 C.

EXAMPLE 2 Preparation of 5,7 n'aphthalenedisalfonic acid 3 (P-hydroxyphenylazo)-p-t0laene sulfonate, disodium salt 9.77 g. of the azocompound derived from coupling diazotized amino G acid into phenol bythe known procedure was dissolved in 250 ml. of water containing 0.92 g.of sodium hydroxide and 2.23 g. of sodium carbonate. The temperature wasraised to 40 C., and there was added with stirring a solution of 4.19 g.of p-toluene-sulfonyl chloride in 20 ml. of acetone. The solution was"stirred at 40-45 for one hour, then raised to 50 and clean sodiumchloride added to precipitate the product. The product was collected byfiltration and washed with 200 ml. of 20% brine solution. Purificationof the product was achieved by dissolution in dimethylformamide atambient temperature, filtration and recrystallization from acetone.

EXAMPLE 3 Preparation of 5,7-naphthalenedisulfonic acid, 3-(alpha 4dihydroxy-m-tolylazo)-ar-s0dium derivative, disodium salt 91 g. of aminoG acid was diazotized with hydrochloric acid and sodium nitrite in theusual manner. The cold diazonium slurry was added at ambient temperatureto a EXAMPLE 4 Preparation of 5,7-naphthalenedisulfonic acid-S-[4-(sulfomethylamino)-0-t0lylaz0], trisodium salt 42.4 g. of 71.6% amino Gacid was dispersed in 600 ml.

of H 0 and 25 ml. of 0.3 N hydrochloric acid. This mixture was chilledto +2 C. and 14 ml. of 40% sodium nitrite sodium was added thereto andthe mixture stirred for one hour. Excess nitrite was destroyed and asolution of 68 g. of sodium acetate trihydrate in 400 ml. of water wasadded to the mixture. This was followed by the addition of 24.5 g. ofm-toluidine omega salt and the mixture was stirred for one hour. ml. of5 N sodium hydroxide and 20 g. of sodium chloride were then added. Themixture was placed in an ice bath and stirred for one hour. The productprecipitated, was filtered, washed with 30% brine and dried-at 60 C.77.3 g. of product was recovered.

8 EXAMPLE 5 Preparation of 5,7-naphthalenedisulfonic acid, 3-(2,4,6-

trihydroxy-S-pyrimidinylazo) -dis0diam salt 18.9 g. of amino G acid wasslurried in 150 ml. of water and ice at 5 C. 20 ml. of 5 N hydrochloricacid was added, followed by dropwise addition during V2 hour of 12.5 ml.of 4 N sodium nitrite. The Whole was stirred at 5 C. for 15 minutes andexcess N0 was discharged by addition of sulfamic acid. The resultingdiazo slurry was added slowly to a well-stirred slurry of 7 g. ofbarbituric acid in 150 ml. of H 0 and ml. of 20 g./100 m1. Na COsolution at 10 C. keeping the temperature below 10 C. and the whole atpH 8-9. The resulting bright yellow slurry was stirred in 600 ml. ofboiling 10% brine and filtered hot. The whole was chilled to roomtemperature and filtered. The cake was washed with 50 ml. of 10% brineand dried at 75 C. for 18 hours; yield 30.08 g.

EXAMPLE 6 Preparation of 3-naphth0l-7-sulf0nic acid2-[(rn[(4,6-diamino-s-triazin-Z-yl) amino] phenyl) carbam0yl]-4-(2-methoxy-S-salfophenylazo) 2.03 g. of 4-methoxy metanilic acid wasdissolved in 50 ml. of water and ice. 6 ml. of hydrochloric acid wasadded, followed by 0.7 g. of sodium nitrite in water. The whole wasstirred for 15 minutes and a small amount of sulfamic acid added. Theresulting cold solution was then slowly added (5 minutes) to awell-stirred solution of 4.67 g. of2-[(m-[(4,6-diamo-s-triazin-Z-yl)amino]- phenyl)carbamoyl]-3-naphthol-7-sulfonic acid dissolved in 100 ml. of water and10 ml. of 5 N sodium hydroxide. The whole was stirred for 1 /2 hours atpH 11 and filtered with filter aid present. The filtrate was made acidto Congo Red with 5 N hydrochloric acid and the precipitate collected byfiltration.

The solid was dissolved in ml. of boiling water with sodium hydroxide toa positive phenolphthalein end point. The whole was filtered and 20 g.of sodium chloride added to the hot solution. The mixture was stirred atroom temperature and the precipitate collected by filtration and washedwith 25 ml. of 7.5% brine and dried at 75 C. in a vacuum for 18 hours;yield 5.28 g.

EXAMPLE 7 Preparation of 3-naphthol-7-salf0nic acid, Z-carbamoyl-4-(Z-methoxy-5-salfophenylaz0) 10.2 g. of 2-amino-4-sulfoanisole wasdissolved in 100 ml. of water and 30 ml. of 5 N hydrochloric acid byheating the mixture close to the boiling point. The reaction mixture wascooled to room temperature and the solution then brought to 5 C. withice. 50 m1. of 1 N sodium nitrite was added slowly to form a diazosolution. After 15 minutes of stirring a small amount of excess N0 wasdischarged by addition of sulfamic acid. The diazo solution was added toa filtered solution of 69.2 g. of 2-hydroxy-1,6-disulfo-3-naphthoicamide dissolved in 300 m1. of water with 30 ml. of sodium hydroxide and20.4 g. of sodium acetate monohydrate. After dissolving and filtrationof the coupling compound, hydrochloric acid was added to 10 C. until thereaction mixture was acid to Congo Red. The pH was slowly raised to 6.5-7.0 by the addition of 50 ml. of 5% sodium bicarbonate and 20 ml. of 20%sodium carbonate. The whole was stirred at 20 C. for 2 hours. The brightred dye slowly began to precipitate. 90 g. of sodium chloride was addedand the mixture stirred for 1 /2 hours. The whole was filtered and thecake washed with 50 ml. of 10% brine. The cake was dissolved in 200 ml.of boiling 10% brine and filtered using a filter aid. The filtrate wascooled to room temperature and the precipitate collected. The cake waswashed with 30 ml. of 10% brine and dried at 70 C. for 2 days; yield15.9 g.

9 EXAMPLE 8 Preparation of carbzmilic acid,4-(5,7-disulf0-3-naphthylaz)-3-methyl-, I-ethyl ester, disodium salt22.3 g. of direct yellow RFL base was dissolved in 150 ml. of 0.33 Nsodium hydroxide. 10.8 g. of ethyl chloro formate dissolved in 75 ml. ofether was added dropwise in 1 hour with stirring at room temperature.The mixture was stirred for 19 hours and the water layer extracted withether. The ether extract was poured into acetone, a precipitate forming,which was then digested on the steam bath. This mixture was thenfiltered and washed with acetone and ether; yield 22.5 g. The productwas dissolved in dimethylformamide, filtered and reprecipitated byadding to acetone.

EXAMPLE 9 Preparation of 5,7-naphthalenedisalfonic acid, 3-[4-([5-ethylhexahydro 3 (hydroxymethyl) 2 0x0 s triazin-l-yl] methyl)amino-o-tolylaza], disodium salt EXAMPLE 11 Preparation ofS-(Z-hydmxyphenylazo)-6-hydr0xynaphthalene-2,7-disulfonic acid 327 g. ofo-amino phenol was mixed with 200 cc. of water, g. of sodium chloride,300 cc. of 0.3 N sodium nitrite, and 75 cc. of 10 N hydrochloric acid. Asolution of 114 g. of R-Salt in 1 liter of water and 106 g. of sodiumcarbonate was then prepared. At room temperature the o-amino phenolmixture was added to the R- N=N -NH, Boom-N N-CHzOH NaO;S L

OgH

MW 465.4 MW 189.2

A solution was prepared containing 20.8 g. of Aerotex Resin 28solids=10.4 g.=.055 mole) and 38.2 g. (61% real=.050 mole) of directyellow RFL base in 125 ml. of water. The pH (8.8) was adjusted to 7.1with N/l HCl. The mixture was then stirred at 75-80. until a nearlynegative coupling test was reached (2 /2 hours). After standingovernight at room temperature, a reaction mixture was reheated to 75 andtreated with sodium chloride. The product obtained on cooling wasfiltered off, washed with acetone and ether and dried under vacuum. Thered-orange solid amounted to 40.4 g. On the salt-free basis(NaCl=25.8%), the yield was 88%.

EXAMPLE 10 Preparation of3-hydroxy-4-(2-salfophenylaz0)naphthalene-7-sulfonic acid 52 g. oforthanilic acid was dissolved in 0.3 N sodium hydroxide, 100 cc. ofWater and 3 cc. of 0.3 N sodium nitrite. This mixture was then pouredinto 100 cc. of 10 N hydrochloric acid and ice. 89.3 g. of Schaifer Saltwas almost dissolved in 12.5 cc. of water at room temperature. To thismixture was added 106 g. of sodium carbonate. The orthanilic acidmixture was then slowly added to this mixture with constant stirring andstirring continued for one hour. After one hour the mixture was heatedto 90 C., 300 g. of sodium acetate trihydrate was added NaOrS- Saltsolution with constant stirring. After diazotization was complete, 900g. of sodium acetate trihydrate was added to the mixture and the mixturewas allowed to stand overnight. A spongy mass formed which was washedwith a 20% solution of sodium acetate trihydrate to remove excess sodiumcarbonate and R-salt. The recovered product was dried at 70 C. C.; yield142 g.

EXAMPLE 12 below was fed to the chicks ad libitum for 3 hours. Only.

the control diet contained no potentiating compound. Six chicks wereused in each treatment. Immediately following the 3-hour feeding period,the chicks were bled by cardiac puncture. each bird and assayedmicrobiologically. An average of the results obtained with each group ofchicks is reported in Table 1 below.

1% ml. of blood was taken from TABLE 1 Percent 200 pp in Blood CTC ByWeight Supplement OTC a s mcgJml. Supplement CTC HCl Control-0-...-. 0OTC 1 0.127 0.5 8-amino-l-naphthol-5,7-disulfonic acid CTC .325 0.53-hydroxy-4-phenylazo-5,7-naphthalenedi- OTC .260

sulfonic acid. 0.5 5,7-naphtha1enedisulfonic acid, 3[4([5- OTC .237

ethylhexa-hydro-B-(hydroxymcthyl)- 2-0x0-s-triazln-1-y1]methy1)amino-otolylazo] disodium salt. 5,7-naphthalenedisulfonic acid,3-(2,4,6- CTC .234

trilltiydroxy-5-pyrlmidinylazo) disodium sa 5,7-naphthalenedisulfonicacid, 3-(a,4' OTC .280

dihydroxy-m-tolylazo)-ar-sodium derivative, disodium salt.5,7-naphthalenedisulfonic acid, 3-an1ino OTC .340

disodium salt. 3-naphthol-5,7 disulfonic acid OTC .2275,7-naphthalenedisulfonic acid, 3-(4- OTC .250

acrylamido-o-tolylazo) disodium salt. LEI-naphthalene disulfonic acidOTC .207 5,7-naphthalenedisulionic acid, 3-(4-amino- OTC .227

o-tolylazo) disodium salt. 8-amino-5,7-naphthalene disulfonic acid OTC.193 Carbanilic acid, 4-(5,7-dlsulfo-3- OTC .167

naphthylazo)-3-methyl, l-ethyl ester, disodium salt.5,7naphthalenedisulfonic acid-B-(p- CTC .153

methylaminophenylazo) disodium salt. 5,7-naphthalencdisulfonicacid-2-[m(m- CTC .220

nigobenzamido) benzamido] disodium sa 3-arnino-5,7-naphthalenedisulfonicacid .160

hydrochloride. 0.5 6,7-naphthalenedisu1ionic acid-s-[4- .177

(sullomethylamino)-o-tolylazo] trlsodium s C0ntrol-0. 0 OTC i .1350.5.-- 8amino-1-naphth0l-5J-disulfonic acid CTO 2 .280 0.53-naphtho1-7-sulionic acid, 2-carbamoyl-4- OTC .190

(Z-methoxy-5-sulfophenylazo) 0.5 3-nephth01-7-sulionic acid-2-[(m-[(4,6-OTC .177

dicmino-s-triazin-fl-yl) amino] phenyl) carbam0yl]-4(Z-methoxy-fi-sulfophenylazo). 2-naphthol-6-sulionlc acid, 3[(m-amino-CIO .183

phcnyl) carbamoyl]. 3-naphthol-7-sulfonic acid-2-[(m-[(4,6- OTC .160

diamino-s-triazln-Zyl) amino] phcnyl) carbarnoyl].2,7-naphthalenedisulionic acid-4,5- OTC .180

dihydroxy-3-(6-methoxy-4-nitro-mtolylazo). 4-naphthol-2,7-disulfonicacid, 5arnino-3-(4 OTC .153

chloro-6-sulfo-3-tolylazo) trisodium salt.5-amino-4-hydroxy-6-(4-nitr0phenylaz0)-3- CTC .223

plielnylazoafl-naphthalene disulfonic ac disodium8-amino-1-hydr0xy-7-(8-nitro- OTC .210

phenylazo)-2-pheny1az0naphtha1ene-3,6- disulfonate.1,5-naphthalenedisulfonic acid-7-[p-(p- OTC .263

anlignobenzamido)-benzamido] disodium sa 0 GT0 .075S-amino-l-naphthol-5,7-disullonic acid OTC .210S-hydroxy-4-(2-su1fophenylazo) naph- OTC .240

thalene-7-sullonic acid. 0 GT0 .058 8-amino-1-naphtho1-5,7-disu1fonicacid OTC .160 5-(m-tolylazo)-6-hydroxynaphthalene-2,7- OTC .086

disulfonic acid. 5-(2-hydroxyphenylazo)-6-hydroxynaph- OTC .093

thalene-2,7-disulfonic acid.

1 Average of 9 tests. Average of 6 tests.

EXAMPLE 13 It will be noted from the results in Table 1 above, that inall instances where the potentiating agents were incorporated in a dietcontaining chlortetracycline, increase 7 in blood levels ofchlortetracycline occurred. In many instances the blood levels ofchlortetracycline doubled and in others it was found to be nearly threetimes that obtained with the control diet which contained nopotentiating agent.

Following the procedure set forth in Example 12, groups of six chickswere fed a commercial 'broiler feed diet containing 0.8% calcium ascalcium carbonate, supplemented with 500 p.p.m. of a selectedtetracycline antibiotic and about 0.5% of a potentiating agent. Controltests were run with diets containing 500 p.p.m. of the selectedantibiotic but no potentiating 'agent. The results of these testsarereproduced in Table 2 below.

TABLE 2 500 p.p.m. antibiotic Percent Supplement by Weight Blood BloodBlood Blood Supplement T0 DMCTC 1 TC 1 OTC 1 rncgJml. mcg./ml. mcgJml.mcgJml Control-0 0 0.17 .17 .17 .13 0. 8-amino-l-naphthol-5,7-disullonicacid 0.38 .27 .27 .25 'lPA 0.28 .18 .20 .21 8-arrniio-l-naphthol-5,7-disulionic acid+ 0.59 .57 .43 .28S-amino-I-naphthol-5,7-disulfonlc acid 0 .40 .33 .28 .26 TP A 0 .29 .25.25 .21

1 GT0 Chlortetracycline.

DMC T C D emethylchlortetraoycline. OTC Oxytetracycline.

TPA =Terephthalic acid.

TC =Tetracycline.

The results shown in Table 2 clearly establish the potentiating effectof the compounds of the subject invention with respect to thetetracycline antibiotics. Moreover, they establish that certaincompounds of the subject invention increase blood levels of antibioticconsiderably above those [levels obtainable with known potentiators suchas terephthalic acid. Additionally, they show that with knownpotentiators, such as terephthalic acid, blood levels from a givenamount of a tetracycline antiobiot-ic can not be significantly increasedabove that which is obtainable with the optimum dosage of suchpotentiator. Even at approximately twice the optimum dosage, bloodlevels of antibiotic remain essentially un- A pulverized corn dietcontaining 1% calcium as calcium carbonate and 200 ppm. ofchlortctracycline as the hydrochloride salt thereof was prepared. Tothis diet was added .5% by weight of the potentiator to be tested. Themixture was placed in stainless steel containers and tumbled for /2 hourto assure thorough mixing. The test diets thus prepared were then fed:ad libitum. for three hours to groups of five-weekold chicks which hadbeen starved overnight. After 3 hours feeding time the chicks were bledby cardiac puncture and the blood biologically assayed forchlortetracycline.

The results obtained were similar to those obtained in theabove-mentioned tests, and the results are reported changed. 35

Finally, and perhaps most importantly, the results in m Table 3 below.

TABLE 3 8-arnino-1- Diet CaCO; TPA 1 0.5% naphthol-5,7- EDTA 1 Blood CTCResponse Percent Ca disulfonlc 0.5% mcgJml.

acid 0.5%

l TPA=Terephthalic acid.

EDTA=Ethylenediamine tetra-acetic acid.

Dietpulverized corn.

CTC200 p.p.m. from CTCJICI.

Table 2 clearly establish that the compounds can be used in combinationwith known poten-tiating agents to increase the blood levels lOfantibiotic far above those obtainable with such agents even when suchagents are employed at dosage levels far exceeding their optimum levels.This discovery suggests that the physiological mechanism which producesincreased antibiotic blood levels with known potentiating agents such asterephthalic acid is quite different from that mechanism which producesin creased antibiotic blood levels with the compounds of the subjectinvention. While the exact mechanism is not known, it is thought thatcompounds of the subject invention increase the blood level oftetracycline antibiotics by increasing intestinal absorption. It isbelieved that potentiating effect of compounds such as tetrephthalicacid increase the blood level of the tetracycline antibiotics byreducing kidney excretion 'of the antibiotic.

EXAMPLE 14 To further illustrate the beneficial effects produced by thenovel adjuvants in combination with known potentiating agents, thefollowing tests were conducted.

The following tests illustrate the increased bactericidal or therapeuticactivity of chlortet-racycline produced by the incorporation of apotentiatin-g agent in a poultry diet containing chlortetracycline.

EXAMPLE 15 A broiler feed diet of the formulation set forth below wasemployed.

Vitamin A+D, dry 2,000 u./ig. D (Nopcay) 10,000 u./g. A Profactor-B,mtg/ton Two types of control tests were run simultaneously with thetests for increased activity produced by incorporation of aipotentiating agent in the feed. In one control, the above dietcontained no medication. In the other, .0125 by weight ofchlortetracycline was added as a supplement to the diet. In the testingfor increased activity, .3% by weight ofS-amino-lnap!hthol-5,7-disultonic acid and .0125% by weight ofchlortetracycline was added to the diet.

Diets, medicated and unmedicated, were presented to equal weight groupsof ten chicks each, two days prior to oral inoculation with 5000sporulated oocysts of Eimeria acervulina (etiological agent for one formof intestinal coccidiosis in chickens) and a suflicient number ofsporulated oocysts of Eimeria tenella (etiological agent for cecalcoccidiosis in chickens) to produce about 80% mortality in the untreatedcontrol groups. They were permitted feed and water ad libitum.

Two days after presentation of the feed sporulated oocysts of E. tenellaand E. acervulina were inoculated directly into the crops of all of thebirds in the test by means of a plastic tube attached to a syringe. Onthe fourth day after inoculation group weights were taken and from thesethe average percent weight gain was calculated. The test was terminatedon the eighth day after inoculation. Birds in active groups were againweighed to determine percent weight gain.

The results of these tests are reported in Table 4 below and from theseresults it can be seen that a noticeable increase in the activity of theantibiotic was achieved with incorporation of a potentiating agent inthe diet.

fected rand uninfected controls were run simultaneously with test groupsadministered -a variety of medicated feeds. The basic diet employed wasthe same as that employed in Example 15 above and the type and quantityof medication added to the diet can be readily determined trom Table 5below.

TABLE 5 P.p.m. of 8-amino- No. Alive/Total P.1J.m.of OTC l-naphthol-S,7- P.D.m. 0! TPA No. 7 days after Added to Diet disultonic acid Added toDiet Inoculation addedto diet 1 Uninfected. Infected.

From these results it will be noted that the addition "of 3000 p.p.m. of8-amino-1-naphthol-5,7-disulfonic acid to a feed containing 125 p.p.m.of chlortetracycline, more than doubled the activity of .thechlortetracycline against E. tenella. It may also be noted that thecombination of 3000 p.p.m. of 8-amino-1-n-aphthol-5,7-disulfonic acid,5000 p.p.m. of terephthalic acid and 60 p.p.m. of chlortetracycline wasmore than twice as effective as 125 p.p.m. of chlortetracycline alone.

EXAMPLE 17 The novel adjuvants in combination with chlortetracyclinewere also found to be effective against Salmonella gallinarwm in chicks.In the following tests chicks infected with the above organism were feda standardbroiler-feed diet, as described in Example 12, containingvarious quantities of chlortetracycline in combination with varyingamounts of 8-amino-1-naphthol-5,7-disul- TABLE 4 Alive/Total PercentWeight Percent Weight Diet Drug at end of test Gain 4th day Gain 8th dayControl test as described above. 1/10 D0 3/10 38 5/10 59 66 7/10 56 64TC 3/10 60 88 .3% 8-amin0-1-naphth0l-5,7 disulionic acid plus 9/10 63 82.011% GT0. .3% 8-amino-1-nap11thol-5,7 disulionic acid plus 9/10 65 920125% OTC. .3% 8-amino-1-napl1thol-5,7 dlsulfonic acid plus 10/10 72 84.0125% OTC.

Adding .3% of a potentiating agent to the diet con- 55 ionic acid. Theresults of these tests are provided in taining chlortetracycline doubledthe activity of the chlortetracycline against E. tenella. This isevidenced by the fact that only eight of the twenty birds treated withchlortetracycline alone in the diet were still alive at the end of thetest .period, whereas twenty-eight of the thirty birds treated withchlortetracycline and the potentiating agent were alive at the end ofthe test period. It should also be noted that increasing theconcentration of chlortetracycline from 011% to .02Q.% in a dietcontaining chlortetracycline as the only medication reduced the numberof deaths in groups of 10 chicks from 5 to 3; whereas, employing .011%chlortetracycline and supplementing this with .3% of the p-otentiatingagent reduced the number of deaths in groups of '10 chicks from 5 to 1.

EXAMPLE 16 To further illustrate the effectiveness of the combination ofthe novel potential-tors with chlortetracycline and terephthalic acidagainst disease, tests were run according to the procedure set forth inExample 15 above. In.-

Table 6 below.

TABLE 6 [Efiectiveness against S. gallz'namm, all chicks infected] Thecombination of chlortetracycline and adjuvant were also found to beeifective against Mycoplasma gallisepticum (PPLO)-in chicks. Noticeableincrease of activity was recorded when 200 p.p.m. of chlorotetracyclineand 0.24%, by Weight, of 8-amino-1-naphthol-5,7-

disulfonic acid were incorporated in a standard broiler feed biotic andfrom about 1 to parts by weight per 1,000

parts by weight of the feed of a compound of the formula If: If:

1 l SOSR R5 wherein R is a member of the group consisting of H andalkali metal; R is a member of the group consisting of H and NH R is amember of the group consisting of H and OH; R is a member of the groupconsisting of H and R is a member of the group consisting of H, NH OH,

and -N=NZ wherein Z is a member of the group consisting of phenyl,

N gHg @CEBOH ONa NHCH;

N H C aSOsNa and R is a member of the group consisting of H andphenylazo.

2. An animal feed composition according to claim 1 in which thetetracycline antibiotic is chlortetracycline.

3. An animal feed composition according to claim 1 which also containsterephthalic acid.

4. An animal feed composition effective in accelerating the growth rateand combating disease of non-ruminants which comprises anutritionally-balanced animal feed to which is added from about 50 to1000 parts per million based on the weight of the feed of a tetracyclineantibiotic and from about 1 to 5 parts by weight per 1000 parts byweight of the feed of a compound of the group consisting of and8-amino-l-naphthol-5,7-disulfon-ic acid;

3-hydroXy-4-phenylazo-5,7-naphthalenedisulfonic acid;

5,7-naphthalenedisulfonic 'acid, 3-[4-([5-ethylhexahydro-3-(:hydroxymethyl) 2-oxo s-triazin-l-yl]methyl) aminoo-tolylazo]disodium salt;

5,7-naphthalenedisulfonic acid, 3-(2,4,6-trihydroxy-5- pyrimidinylazodisodium salt;

5,7-naphthalenedisul-fonic lacid, 3(a-4dihydnoxy-mtoly'lazo-ar-sodiumderivative, disodium salt;

5,7-naphthalenedisulfonic acid, 3-'amino disodium salt;

3-naphthol-5,7 disulfonic acid;

5,7-naphthalenedisulfonic acid, 3-(4 acrylamido-0- tolylazo) disodiumsalt;

1,3-naphthalene disulfonic acid;

5,7-naphthalenedisulfonic acid, 3-(4-amino-ot-olylazo disodium salt;

8-amino-5,7-naphthalene disulfonic acid;

carbanilic acid, 4-(5,7-disulfo-3-naphthylazo)-3-methy1,

l-ethyl ester, disodium salt;

5,7-naphthalenedisulfonic acid-3- (p-methylaminophenylazo) disodiumsalt;

5,7-naphtha1enedisulfonic acid-2-[m(m-nitrobenzamido) benzamido]disodium salt;

3-amino-5,7-naphthalenedisulfonic acid hydrochloride;

5,7-naphthale-nedisulfonic acid-s-[4-(sulfomethylamino)- o-tolylazo]trisodiurn salt;

3-naphthol-7 sulfonic acid, 2-carbarnoyl-4- (Z-methoxy-S-sulfophenylazo)

1. AN ANIMAL FEED COMPOSITION EFFECTIVE IN ACCELERATING THE GROWTH RATEAND COMBATING DISEASE OF NON-RUMINANTS WHICH COMPRISES ANUTRITIONALLY-BALANCED ANIMAL FEED TO WHICH AS ADDED FROM ABOUT 50 TO1000 PARTS PER MILLION BASED ON THE WEITHT OF THE FEED OF A TETRACYCLINEANTIBIOTIC AND FROM ABOUT 1 TO 5 PARTS BY WEIGHT PER 1.000 PARTS BYWEIGHT OF THE FEED OF A COMPOUND OF THE FORMULA